Fluorescence Microscopy of Single Molecules

In the Laboratory edited by

Topics in Chemical Instrumentation

David Treichel Nebraska Wesleyan University Lincoln, NE 68504

Fluorescence Microscopy of Single Molecules Jan Zimmermann, Arthur van Dorp, and Alois Renn* Physical Chemistry Laboratory, Swiss Federal Institute of Technology, ETH Hoenggerberg, CH 8093 Zürich, Switzerland; *[email protected]

Single-molecule spectroscopy and detection has matured from a scientist’s dream (1) to a powerful technique widely used in basic and applied science (2) and nowadays is regarded as a bottom-up approach in nanotechnology (3, 4). Extremely sensitive detection techniques such as laser-induced fluorescence allow the study of physical, chemical, and even biological processes (5, 6) on a molecular scale. At present, experiments based on single-molecule detection are starting to enter the field of chemical and physical education (7). One of the preconditions to detect single molecules at room temperature—to make them spatially distinguishable—is achieved by the ability of the microscopy to look at extremely small (diffraction limited) spots. While confocal microscopes have become work horses in single-molecule detection (8), wide-field instruments are especially suited to investigate a large number of molecules in parallel and to bridge singlemolecule data and ensemble measurements. The signal in a single-molecule experiment stems from a repetitive excitation–emission cycle. For common fluorophores, the optical excitation–emission cycle rates are as large as 108 per second with a few microwatts of laser power focused to a diffraction limited spot. With the collection efficiencies of high numerical aperture microscope objectives (up to 30%) the signals of single molecules can be detected with an excellent signalto-background ratio. The temporal dynamics of photon emission of single molecules can be analyzed on a variety of timescales and provides insight into the photophysics and photochemistry of a chemical species and its local environment. On a very short timescale (ns), the photon stream emitted by a molecule exhibits so-called antibunching (9), which is caused by the ability of a single molecule to emit only one photon at a time, and thus regarded as a typical signature of a single quantum emitter. On a longer timescale, a molecule may undergo intersystem crossing into a long-lived triplet state. These triplet excursions virtually separate the emitted photon stream into bright (on) and dark (off ) periods, also a typical behavior of a single quantum system. This triplet blinking has been intensively investigated at liquid helium temperature (10), and recently experiments under ambient conditions have been reported (9, 11, 12). Clearly, for single-molecule detection, the intersystem crossing rate should be as low as possible, and when such a transition occurs, the duration of the dark periods, that is, the triplet state lifetime, should be short. It is well known that oxygen strongly quenches excited states— most prominently the long-living triplet states—thereby opening up a possibility to directly manipulate fluorescence. In this article we describe a wide-field fluorescence microscopy approach that allows students to observe fluorescing single molecules in real time and thereby the investigation www.JCE.DivCHED.org

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of photochemistry and photophysics of individual quantum systems. With the single-molecule detection setup described here, students will be able to directly visualize and analyze such effects and thereby obtain a firsthand impression of the properties of single quantum systems. Experimental Setup The setup consists of a lab-built inverted microscope and is schematically illustrated in Figure 1. All components of the instrument are easily accessible and can be assembled and operated by students in an advanced physical or physicochemical laboratory course. The apparatus has proven to be

Figure 1. Schematics of the experimental setup (top view) used for single-molecule detection. The microscope part is illustrated in more detail (side view). The mounts of the dichroic mirror and the mirror below the microscope objective are placed on translation stages to facilitate translational alignment of the laser beam when entering the microscope.

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(460P, Newport Corp.) is precisely adjustable with piezoelectrically driven MRAs (8322, New Focus). A drop of immersion oil with low autofluorescence is placed between the microscope slide and the objective to achieve index matching of the high NA objective. The fluorescence light is collected by the same microscope and passes the dichroic mirror. Residual laser stray light is blocked by a Raman cutoff filter (XR3002, Omega Optics) and a holographic notch filter (HNPF 532, Kaiser Optical Systems, Inc.). After focusing by a zoom objective (Nikon Nikkor 1:4, 75–200 mm), the expanded (40–100×) fluorescence images are recorded by a CCD camera (Sensicam QE, PCO) connected to a PC. The high resolution chip (1376 × 1040 pixel, size 6.45 × 6.45 µm) has a quantum efficiency greater than 60% and low dark noise is provided by Peltier cooling of the detector. With an AD conversion factor of 2 electrons per count, and assuming a collection efficiency of 10%, approximately 30 photons emitted from a molecule generate a count in one of the pixels. At the CCD chip the fluorescence spot of a single molecule appears distributed over a range of 30 µm (5 pixels, fwhm). Taking into account an expansion factor of 100, this corresponds to a spot size of 320 nm at the sample, which is close to the diffraction limit. The lipophilic tracer DiI (1,1'-dioctadecyl-3,3,3',3'tetramethylindocarbocyanine perchlorate, Molecular Probes) was used as fluorescent dye. The maximum extinction coefficient (εmax ∼1.5 × 105 L mol᎑1 cm᎑1 at 549 nm) corresponds to a molecular absorption cross section of 2.5 × 10᎑16 cm2, which results in an excitation rate of the order of 106 s᎑1 at an intensity of 1 kWcm2. A convenient sample consists of a thin polymer film (poly(methyl methacrylate); PMMA) with DiI coated on a microscope cover slide by means of a lab-built spin coater (∼3000 rpm). The samples were prepared using a 10᎑9 M solution of DiI in toluene with 3% (ww) PMMA. A single drop of solution was applied to the fast spinning cover slide to obtain a thin film with a uniform distribution of dye molecules (thickness ∼50 nm). All preparations

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a straightforward and convenient means to reach single-molecule sensitivity under wide-field operation. Additionally, the setup can be used in a classroom demonstration experiment to visualize properties of single molecules. The light of a frequency doubled Nd YAG laser (CrystaLaser, GCL025L) is directed via a dichroic mirror (560 DRLP, Omega Optics) into a microscope objective (Nikon Plan Fluor 100× NA 1.3) to illuminate the sample. A widefield lens (WL, f = 60 mm) creates a 25-µm diameter spot in the focal plane of the objective. Assuming that all light emitted by the laser (25 mW) is transferred to the sample, a maximum intensity of about 6 kWcm2 is obtained. A microscope cover slide mounted on a XYZ translation stage

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Figure 2. (A) Schematic representation of a 3D data cube generated from a series of sequentially recorded images. The first frame shown gives a translucent image of the data cube displaying the maximum intensities collected throughout the picture series. Different areas can be selected and analyzed. Depending on the size of the selected area a time trace of a fluorescing single molecule (B) or an ensemble of molecules (C) is obtained. The axes are time (s) versus intensity.

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Time / sec Figure 3. Time traces of individual single molecules extracted from a data cube of 100 images, each recorded with 2 s exposure time. All three molecules exhibit a stepwise bleaching process typical for single quantum systems. The detailed process however varies from single step bleaching (solid line) reoccurring or “blinking” behavior (dashed line), to two step bleaching (dotted line). Because all molecules were bleached, the plot was truncated after 90 s.


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can be made at room temperature and ambient pressure in the presence of air. To remove fluorescent impurities, the cover glasses were baked during several hours at 500 ⬚C prior to coating. To immerse the sample in N2, a flexible tube of 2-mm inner diameter is fixed above the sample and connected to a nitrogen gas bottle. A gentle flow of N2 removes the air from the sample surface and, via fast diffusion, the oxygen molecules from the sample. The setup, with exception of the laser, the first mirror, and the wide-field illumination lens, is placed in a black cardboard box to minimize stray light. Hazards The chemicals used here require standard precautions usually met in laboratory experiments. To our knowledge, the hazards of DiI have not been thoroughly investigated. We recommend handling with the same caution as other laser dyes. Avoid exposure of skin and inhalation. Toluene is highly flammable. Also avoid inhalation. Avoid contact of PMMA with the skin and the eyes. Proper eye protection equipment against laser radiation should be worn. Results The illuminated spot of 25 µm was detected in a software selected region of 384 × 384 pixels. Bright spots distributed in the field of view are assigned to be single molecules. To investigate the time dependence of molecular fluorescence, a series of images was recorded with appropriate exposure time. The images were stored on a PC and evaluated by lab-written routines based on a data visualization software package (PV wave, Visual Numerics). First, a virtual data cube as indicated in Figure 2A is constructed from a series of sequentially recorded images. The front view of the cube shows a translucent image of the xy plane of the data cube, that is, the maximum of all intensities collected throughout the image series along the time axis. Also shown is the basic evaluation procedure of the experiment: in the xy plane of the data cube, regions of integration can be de-

fined. The intensities in a selected area are added and the result is plotted along the t axis. Figures 2B and 2C show the typical intensity versus time traces: a small integration region (B) that contains a single spot in the xy plane—a single molecule—and a large area of integration (C) representing an ensemble of molecules. In contrast to the smooth (almost) exponential decay as shown in Figure 2C for the large number of molecules, the individual molecules exhibit a variety of different behaviors. Three traces of single molecules extracted from a data cube of 100 frames taken at 2 s integration time per image is shown in Figure 3. The traces show a stepwise bleaching behavior, a typical signature of single molecules. The first molecule (solid or red line) shows a single step bleaching after a relatively long irradiation time (∼75 s). The second molecule (dashed or green line) immediately bleaches (2 s) but reappears after an irradiation time of about 70 s. After another 10 s of photon emission, the molecule bleaches again. The third molecule (dotted or violet line) shows a two step bleaching behavior. The first step occurs after 5 s and a final bleaching step after 20 s. The apparent two step bleaching process may originate either from two closely spaced molecules, which makes them nonresolvable for the microscope, or a stepwise change of the orientation of the chromophore. This could turn the transition dipole in a less favorable direction with respect to the polarization of the exciting laser. The reoccurrence of molecules as observed with molecule two, so-called “blinking”, is a characteristic feature occurring in single-molecule detection. The reason for this blinking behavior is unclear and a matter of discussion. In our case a cis–trans isomerization of the DiI molecule from the fluorescing trans isomer to a nonfluorescing cis isomer and back might be a possible explanation (13). In addition to the analysis of the xy plane of a data cube, side views (xt and yt planes) can be used to visualize the temporal behavior of the fluorescing molecules, which appear as bright traces. This allows the coordinative identification of single molecules in the xy plane and greatly improves the selection of “interesting” single molecules.

Figure 4. Front and side views of the data cubes of a series of images of DiI molecules in PMMA film taken at 2 s exposure time. (A) shows the images of a sample area under nitrogen flux, (B) a sample area without nitrogen flux, and (C) an area under alternating nitrogen and air flux. Note how the intensities of the single molecules are increased in the presence of oxygen (i.e., without nitrogen flux), but how their fluorescing lifetimes are drastically decreased at the same time.

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The front and side views of the data cubes for three experiments conducted under nitrogen flux, air flux, and alternating nitrogen and air flux are shown in Figure 4. The image cubes A and B were scaled by their individual fluorescence intensity. Note the fluorescing lifetime differences of the molecules between the experiment with nitrogen flux and the one with air flux. The fluorescence intensity increases in the presence of oxygen whereas the fluorescing time apparently decreases. In the xt and yt planes of the data cube of the alternating nitrogen and air flux experiment, the periodic increase and decrease of the fluorescence intensity of the molecules can be observed with the alternation of nitrogen and air flux.

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Time / sec Figure 5. Time traces of two individual single molecules under alternating nitrogen and air flux taken at 1 s integration time: solid line—intensely fluorescing molecule, dashed line—less intensely fluorescing molecule. The increase of fluorescence intensity under oxygen rich conditions (air flux) is a result of oxygen quenching of the nonradiating triplet state under strong excitation conditions.

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The time traces of two individual molecules under alternating nitrogen and air (oxygen) flow are shown in Figure 5. The drastic increase of fluorescence intensity in an oxygen rich environment is the direct result of oxygen quenching of the (nonradiating) triplet state under strong excitation conditions. In the absence of oxygen (under triplet saturation conditions), the two molecules show approximately the same intensity in a time average. The presence of oxygen alters the saturation conditions, leading to different intensity levels. Whereas the intensely fluorescing molecule bleaches during the first oxygen rich cycle, the other molecule survives for the whole time of the measurement. To analyze the influence of oxygen on the fluorescent behavior of DiI molecules in detail, additional experiments were conducted with a higher time resolution. The time traces of single DiI molecules under nitrogen and air flux recorded at 5 ms exposure time to a total of 200 images are shown in Figure 6. These traces show the effect of oxygen on the fluorescing molecules. While under nitrogen flux the molecules appear at longer integration times as faint, blinking spots, the photon statistics at short integration time becomes dominated by dark periods (characterized by the triplet lifetime) with frequent “bursts” of fluorescent activity. Flushing with (oxygen containing) air leads to a significant shortening of the dark periods, which on a longer timescale appears as a strong increase of fluorescent activity. The increase of intensity in Figure 6B is due to the lack of time resolution that results in the missing of very short dark periods. This happens more often when the triplet state lifetime is reduced by oxygen. Once experimental conditions favoring single-molecule detection are satisfied, a number of criteria have to be met to ascertain whether the observed signal actually comes from a single emitter. These criteria are: (i) The observed density of emitters is compatible with the known concentration of individual molecules and scales with the original bulk concentration. (ii) The observed fluorescence intensity level is consistent with that of a single emitting molecule. (iii) Each immobilized emitter has a well-defined absorption or emission dipole (for organic dyes usually linear). (iv) Fluorescence emission exhibits only two levels (on–off behavior due to blinking or photobleaching) over timescales where no changes in the environment are expected. (v) The emitted light exhibits antibunching, which is characteristic of a single quantum emitter. (vi) The spectrum of the molecule corresponds to the bulk emission spectrum.

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Time / (5 ms) Figure 6. Time traces of two individual single molecules taken at 5 ms exposure time illustrate the influence of oxygen on the triplet state. In an oxygen free atmosphere (A) the fluorescence emission is interrupted by long breaks (low intensity) characterized by the triplet lifetime. In the presence of oxygen the triplet state is strongly quenched (B), leading to a decrease of the triplet lifetime and thereby a shortening of the dark periods. While on this short time scale the maximum fluorescence intensity remains almost unchanged, longer integration times lead to a virtually more intense signal.

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We have described a convenient and straightforward microscopy setup that allows the observation of fluorescence images of single molecules distributed in a polymer film. By means of this setup, students of advanced physics and chemistry are able to directly observe and experience the behavior of single quantum systems in real time. The experiments described here were performed in a laboratory course consisting of three 3-h lab periods. Additional experiments can be developed that allow a qualitative and quantitative analysis of single-molecule experiments. The polarization of the molecules can be analyzed to give information about molecular orientations (transition dipole moment) and diffusion in vis-


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cous media can be studied. In addition to dye molecules, quantum dots, being representatives of another species of single quantum systems, could also be investigated. Acknowledgments This work was supported by ETH-Zurich. We would like to thank E. Meister for helpful discussions and careful reading of the manuscript. Literature Cited 1. “An atom is a body which cannot be cut in two. A molecule is the smallest possible portion of a particular substance. No one has ever seen or handled a single molecule. Molecular science, therefore, is one of those branches of study which deal with things invisible and imperceptible by our senses, and which cannot be subjected to direct experiment.” Maxwell, J. C. Nature 1873, 8, 437. 2. Single-Molecule Optical Detection, Imaging and Spectroscopy; Basché, T., Moerner, W. E., Orrit, M., Wild, U. P., Eds.; Verlag Chemie: Weinheim, Germany, 1996.

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3. Drexler, K. E. Nanosystems: Molecular Machinery, Manufacturing, and Computation; Wiley Interscience: New York, 1992. 4. Feynman, R. P. There’s plenty of room at the bottom, an invitation to enter a new field of physics. http://www.zyvex.com/ nanotech/feynman.html (accessed Dec 2003). 5. Frontiers in Chemistry: Single molecules. Science (special issue), 1999, 283 (Mar 12). 6. Single Molecule Spectroscopy: Nobel Conference Lectures; Rigler, R., Orrit, M., Basché, T., Eds.; Springer series in Chemical Physics, Vol. 67; Springer Verlag: Berlin, Germany, 2001. 7. Harbron, E. J.; Barbara, P. F. J. Chem. Educ. 2002, 79, 211. 8. Nie, S.; Zare, R. N. Annu. Rev. Biophys. Biomol. Struct. 1997, 26, 567. 9. Fleury, L.; Segura, J.-M.; Zumofen, G.; Hecht, B.; Wild, U. P. Phys. Rev. Lett. 2000, 84, 1148. 10. Basché, T.; Kummer, S.; Bräuchle, C. Nature 1995, 373, 132. 11. Veerman, J. A.; Garcia-Parajo, M. F.; Kuipers, L.; van Hulst, N. F. Phys. Rev. Lett. 1999, 83, 2155. 12. Hübner, C. G. H.; Renn, A.; Renge, I.; Wild, U. P. J. Chem. Phys. 2001, 115, 9619. 13. Widengren, J.; Seidel, C. A. M. Phys. Chem. Chem. Phys. 2000, 2, 3435.


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Fluorescence Microscopy of Single Molecules

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